In 1921, Prausnitz and Kustner documented the presence of a transferable skin sensitizing factor in the serum of allergic patients and laid the cornerstone for the modern concepts of allergy. This serum factor was designated as reagin. The P-K transfer reaction became the essential investigative tool for the study of allergic disorders. In 1966, Ishizaka and colleagues linked the reaginic activity to antibodies belonging to the IgE class of immunoglobulins. Radioimmunoassay procedures were developed for the assay of serum level of total IgE antibodies and of allergen-specific IgE antibodies (the RAST test). It is clear that the RAST response for a given serum actually represents the sum of titers and avidities of the most of IgE antibodies with specificity for the various allergenic components of an allergen extract.
In the clinical practice of allergy, allergen extracts are used both in diagnosis and treatment. The extracts vary considerably in their potency as manifested by their ability to evoke a positive reaction in the human skin. Not unexpectedly, such variability in potency of different batches of extracts prepared by different manufacturers and even in different batches of extracts prepared by the same manufacturer has seriously hindered efforts to standardize allergy diagnosis, specific immunotherapy with injection with allergen extracts, and the in-vitro tests for monitoring the immunologic response to immunotherapy.
The variations in potency of extracts are largely caused by the diverse proteins and other allergenic components in any given allergen extract. The particular component, or more likely components, in any extract which cause the allergic response have not been satisfactorily identified. Fractionation of allergen extracts into their multiple allergenic factors by conventional techniques such as chemical, electrophoretic, isoelectric focusing, etc., would be extremely cumbersome and has not been attempted except on a limited scale.
At present, satisfactory potency standards are not available. Commercial extract preparations carry only an indirect indication of their content of allergenic components in terms of either weight/volume of allergen extract or protein nitrogen unit (PNU), the former being related to Noon units. Since the allergenic components of the extract represent only a small proportion of the extract, it is clear that these indirect indications can deviate from the actual quantity by a factor of several hundred to several thousands.
Several different approaches have been adopted to resolve these difficulties. The feasability of standardization of the potency of extracts by skin testing has been explored on a limited scale but there are several theoretical and practical barriers to this approach. A large number of suitable human volunteers would be required to do the necessary skin testing and there is a substantial risk of local and systemic (possibly fatal) reactions.
Another approach used in the past involves the measurement of the potency of the extract by its ability to inhibit the RAST reaction--the RAST inhibition test. This approach also faces several problems in that the RAST test itself has not been a standardized method. Attempts have been made to resolve this difficulty with the use of a "reference allergen extract" but this itself is a non-standardized preparation. Alternatively, serum containing known titers of allergen-specific IgE antibodies have been used for this purpose.
Since the assay for total IgE antibodies had been standardized with the use of WHO standard IgE material, efforts were made to standardize the RAST test by linking it to the radioimmunoassay procedure for total IgE and creating arbitrary units to express the concentration of allergen specific IgE, but this approach was unsatisfactory. This is because the basic test designs for the immunoassays for total and allergen-specific IgE antibodies are different; the immunosorbent for the total IgE assay is immobilized anti-IgE while that for specific IgE is immobilized allergen. Hence, the binding characteristics in the two systems are not expected to be comparable. FIGS. 1 and 2 illustrate this. Serum levels of allergen-specific IgE antibodies could not be quantitatively estimated as such material in purified form was not available for use as standards in constructing standard curves. Another approach adopted to standardize the results of the RAST test was based on the use of a time control and is designated the Modified RAST test. In this approach, the time required to obtain a given number of counts with a given number of units of WHO standard IgE run with a PRIST disc (anti-human IgE insolubilized on cyanogen bromide activated disc) and RAST isotope was measured and the time determined was used to count the bound radioactivity in the RAST test for all of the test samples. Thus, by varying the counting time, the absolute counts for the Modified RAST time control (and hence the modified RAST cut-off point) were kept constant. However, even this approach will provide only semi-quantitative estimation of the titer of the allergen specific IgE antibodies in the test serum because of a lack of suitable standards.
In addition to the above methods, attempts have been made to define the allergenic components in allergen extracts by gel diffusion precipitation techniques. This has been used on a limited scale for mite extract.
All of the above approaches share one common and serious handicap, namely the basic design of these methods does not include a purification step by which the small quantity of allergenic material (i.e., the multiple allergenic components) in an allergen extract can be separated from the bulk of the proteins and other substances which do not evoke an IgE response. In skin testing and RAST inhibition methods, a large number of substances are present which are of no clinical relevance because they do not induce an IgE response and can introduce error into the test results.
In 1935, it was recognized that specific immunotherapy for inhalent allergy induces the production of "blocking antibodies" which were subsequently found to belong to IgG class of immunoglobulins. Since then a large number of in-vivo and in-vitro methods have been developed to assess IgG response. Among these, the indirect methods include Prausnitz-Kustner neutralization, inhibition of in-vitro release of histamine from allergen-challenged leukocytes, hemagglutination of allergen-coated erythrocytes, and precipitation of IgG antibodies with staphylococcal protein A. The direct methods include radioimmunoassay and enzyme immunoassay. As for immunoassays for allergen-specific IgE antibodies, all the above methods for specific IgG antibodies yield only semi-quantitative results due to lack of standards containing purified material which can be quantified in nanograms or picograms.
The radioimmunoassays and enzyme immunoassay procedures which are being widely used today for the in vitro diagnosis of allergy and in-vivo monitoring of the immunologic responses to specific immunotherapy give only semiquantitative estimation of allergen specific IgE and IgG antibodies in the serum and direct quantitative measurement of these antibodies has not been possible to date. There has been no available technique for producing purified preparations of these antibodies for use as standards in a quantitative assay.
It is accordingly the object of this invention to provide techniques for isolating allergenic components from allergen extracts and for producing purified preparations of allergen specific IgE and IgG antibodies which can be used as standards in quantitative radioimmunoassays and enzyme immunoassays. This and other objects of the invention will become apparent to those skilled in the art from the following detailed description.